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1.
J Biol Chem ; 293(39): 15021-15032, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30087118

RESUMO

N-Formylation of the Met-tRNAMet by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNAMet for protein synthesis in mammalian mitochondria is still debated. Here, we generated a Mtfmt-KO mouse fibroblast cell line and demonstrated that N-formylation of the first methionine via fMet-tRNAMet by MTFMT is not an absolute requirement for initiation of protein synthesis. However, it differentially affected the efficiency of synthesis of mtDNA-coded polypeptides. Lack of methionine N-formylation did not compromise the stability of these individual subunits but had a marked effect on the assembly and stability of the OXPHOS complexes I and IV and on their supercomplexes. In summary, N-formylation is not essential for mitochondrial protein synthesis but is critical for efficient synthesis of several mitochondrially encoded peptides and for OXPHOS complex stability and assembly into supercomplexes.


Assuntos
Hidroximetil e Formil Transferases/genética , Metionina/genética , Mitocôndrias/genética , Biossíntese de Proteínas/genética , Animais , DNA Mitocondrial/genética , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Mutação , Fosforilação Oxidativa , Aminoacil-RNA de Transferência/genética
2.
mBio ; 7(6)2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27834201

RESUMO

YbeY is part of a core set of RNases in Escherichia coli and other bacteria. This highly conserved endoribonuclease has been implicated in several important processes such as 16S rRNA 3' end maturation, 70S ribosome quality control, and regulation of mRNAs and small noncoding RNAs, thereby affecting cellular viability, stress tolerance, and pathogenic and symbiotic behavior of bacteria. Thus, YbeY likely interacts with numerous protein or RNA partners that are involved in various aspects of cellular physiology. Using a bacterial two-hybrid system, we identified several proteins that interact with YbeY, including ribosomal protein S11, the ribosome-associated GTPases Era and Der, YbeZ, and SpoT. In particular, the interaction of YbeY with S11 and Era provides insight into YbeY's involvement in the 16S rRNA maturation process. The three-way association between YbeY, S11, and Era suggests that YbeY is recruited to the ribosome where it could cleave the 17S rRNA precursor endonucleolytically at or near the 3' end maturation site. Analysis of YbeY missense mutants shows that a highly conserved beta-sheet in YbeY-and not amino acids known to be important for YbeY's RNase activity-functions as the interface between YbeY and S11. This protein-interacting interface of YbeY is needed for correct rRNA maturation and stress regulation, as missense mutants show significant phenotypic defects. Additionally, structure-based in silico prediction of putative interactions between YbeY and the Era-30S complex through protein docking agrees well with the in vivo results. IMPORTANCE: Ribosomes are ribonucleoprotein complexes responsible for a key cellular function, protein synthesis. Their assembly is a highly coordinated process of RNA cleavage, RNA posttranscriptional modification, RNA conformational changes, and protein-binding events. Many open questions remain after almost 5 decades of study, including which RNase is responsible for final processing of the 16S rRNA 3' end. The highly conserved RNase YbeY, belonging to a core set of RNases essential in many bacteria, was previously shown to participate in 16S rRNA processing and ribosome quality control. However, detailed mechanistic insight into YbeY's ribosome-associated function has remained elusive. This work provides the first evidence that YbeY is recruited to the ribosome through interaction with proteins involved in ribosome biogenesis (i.e., ribosomal protein S11, Era). In addition, we identified key residues of YbeY involved in the interaction with S11 and propose a possible binding mode of YbeY to the ribosome using in silico docking.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Metaloproteínas/genética , Metaloproteínas/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 16S/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/isolamento & purificação , Proteínas de Ligação ao GTP/metabolismo , Regulação Bacteriana da Expressão Gênica , Simulação de Acoplamento Molecular , Mutação de Sentido Incorreto , Ligação Proteica , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
4.
Mol Microbiol ; 98(6): 1199-221, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26337258

RESUMO

Threonylcarbamoyladenosine (t(6)A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t(6)A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t(6)A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNA(Ile)-lysidine synthetase. We confirm that t(6)A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t(6)A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t(6)A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t(6)A(-) D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t(6)A in tRNAs. Thus, although t(6)A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.


Assuntos
Adenosina/análogos & derivados , Deinococcus/genética , Escherichia coli/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Adenosina/genética , Adenosina/metabolismo , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacilação/genética , Sequência Conservada , Deinococcus/metabolismo , Escherichia coli/metabolismo , Dados de Sequência Molecular , Células Procarióticas , Proteômica , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Saccharomyces cerevisiae/genética
5.
Nat Commun ; 6: 7520, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-26138142

RESUMO

Dominant mutations in five tRNA synthetases cause Charcot-Marie-Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA(Gly) aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Glicina-tRNA Ligase/genética , Neurônios Motores/metabolismo , Movimento , Biossíntese de Proteínas/genética , Células Receptoras Sensoriais/metabolismo , Tirosina-tRNA Ligase/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila , Humanos , Expectativa de Vida , Neurônios Motores/patologia , Mutagênese Sítio-Dirigida , Mutação , Junção Neuromuscular/patologia , Fenótipo , Células Receptoras Sensoriais/patologia
6.
Proc Natl Acad Sci U S A ; 112(19): 6015-20, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25918386

RESUMO

Bacterial strains carrying nonsense suppressor tRNA genes played a crucial role in early work on bacterial and bacterial viral genetics. In eukaryotes as well, suppressor tRNAs have played important roles in the genetic analysis of yeast and worms. Surprisingly, little is known about genetic suppression in archaea, and there has been no characterization of suppressor tRNAs or identification of nonsense mutations in any of the archaeal genes. Here, we show, using the ß-gal gene as a reporter, that amber, ochre, and opal suppressors derived from the serine and tyrosine tRNAs of the archaeon Haloferax volcanii are active in suppression of their corresponding stop codons. Using a promoter for tRNA expression regulated by tryptophan, we also show inducible and regulatable suppression of all three stop codons in H. volcanii. Additionally, transformation of a ΔpyrE2 H. volcanii strain with plasmids carrying the genes for a pyrE2 amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in the pyrE2 gene can be complemented by expression of the amber suppressor tRNA. These results pave the way for generating archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in archaeal and archaeviral genetics. We also provide possible explanations for why suppressor tRNAs have not been identified in archaea.


Assuntos
Archaea/genética , Códon de Terminação , Haloferax volcanii/genética , RNA de Transferência/metabolismo , Supressão Genética , Archaea/metabolismo , Sequência de Bases , Códon sem Sentido , Escherichia coli/metabolismo , Genes Supressores , Haloferax volcanii/metabolismo , Dados de Sequência Molecular , Novobiocina/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Serina/química , Timidina/química , Triptofano/química , Uracila/química , beta-Galactosidase/metabolismo
7.
J Biol Chem ; 289(47): 32729-41, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25288793

RESUMO

N-Formylation of initiator methionyl-tRNA (Met-tRNA(Met)) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA(Met)) for both initiation and elongation. A portion of Met-tRNA(Met) is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.


Assuntos
Hidroximetil e Formil Transferases/genética , Doença de Leigh/genética , Proteínas Mitocondriais/genética , Mutação , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Hidroximetil e Formil Transferases/metabolismo , Immunoblotting , Doença de Leigh/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Biossíntese de Proteínas/genética , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Homologia de Sequência de Aminoácidos , Serina/genética , Serina/metabolismo
8.
PLoS Pathog ; 10(6): e1004175, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24901994

RESUMO

YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing of the critical 3' end of 16 S rRNA and in 70 S ribosome quality control under stress. These central roles account for YbeY's inclusion in the postulated minimal bacterial genome. However, YbeY is not essential in E. coli although loss of ybeY severely sensitizes it to multiple physiological stresses. Here, we show that YbeY is an essential endoribonuclease in Vibrio cholerae and is crucial for virulence, stress regulation, RNA processing and ribosome quality control, and is part of a core set of RNases essential in most representative pathogens. To understand its function, we analyzed the rRNA and ribosome profiles of a V. cholerae strain partially depleted for YbeY and other RNase mutants associated with 16 S rRNA processing; our results demonstrate that YbeY is also crucial for 16 S rRNA 3' end maturation in V. cholerae and that its depletion impedes subunit assembly into 70 S ribosomes. YbeY's importance to V. cholerae pathogenesis was demonstrated by the complete loss of mice colonization and biofilm formation, reduced cholera toxin production, and altered expression levels of virulence-associated small RNAs of a V. cholerae strain partially depleted for YbeY. Notably, the ybeY genes of several distantly related pathogens can fully complement an E. coli ΔybeY strain under various stress conditions, demonstrating the high conservation of YbeY's activity in stress regulation. Taken together, this work provides the first comprehensive exploration of YbeY's physiological role in a human pathogen, showing its conserved function across species in essential cellular processes.


Assuntos
Proteínas de Bactérias/metabolismo , Endorribonucleases/metabolismo , Processamento de Terminações 3' de RNA , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Estresse Fisiológico , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Cólera/enzimologia , Cólera/imunologia , Cólera/metabolismo , Cólera/microbiologia , Toxina da Cólera/biossíntese , Sequência Conservada , Endorribonucleases/química , Endorribonucleases/genética , Regulação Bacteriana da Expressão Gênica , Imunidade nas Mucosas , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Mutação , Filogenia , Vibrio cholerae/imunologia , Vibrio cholerae/patogenicidade , Vibrio cholerae/fisiologia , Virulência , Fatores de Virulência/biossíntese
9.
RNA ; 20(2): 177-88, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24344322

RESUMO

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2(Ile)) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2(Ile) binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.


Assuntos
Anticódon/genética , Haloarcula marismortui/genética , RNA Arqueal/genética , RNA de Transferência de Isoleucina/genética , Uridina/análogos & derivados , Pareamento de Bases , Sequência de Bases , Códon/genética , Escherichia coli/genética , Haloferax/genética , Estrutura Molecular , Mutação Puntual , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA Bacteriano/genética , RNA Fúngico/genética , RNA de Transferência de Isoleucina/química , RNA de Transferência de Isoleucina/metabolismo , Ribossomos/química , Saccharomyces cerevisiae/genética , Sulfolobus/genética , Aminoacilação de RNA de Transferência , Uridina/química , Uridina/genética
10.
Nucleic Acids Res ; 42(3): 1904-15, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24194599

RESUMO

Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA2(Ile) to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNA(Ile)-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA2(Ile) is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA1(Ile), in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.


Assuntos
Bacillus subtilis/genética , Códon , Isoleucina/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Isoleucina/química , RNA de Transferência de Isoleucina/metabolismo , Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/crescimento & desenvolvimento , Deleção de Genes , Fenótipo , RNA de Transferência de Isoleucina/isolamento & purificação , Ribossomos/metabolismo , Aminoacilação de RNA de Transferência
11.
Nat Struct Mol Biol ; 20(5): 641-3, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23542153

RESUMO

Decoding of the AUA isoleucine codon in bacteria and archaea requires modification of a C in the anticodon wobble position of the isoleucine tRNA. Here, we report the crystal structure of the archaeal tRNA2(Ile), which contains the modification agmatidine in its anticodon, in complex with the AUA codon on the 70S ribosome. The structure illustrates how agmatidine confers codon specificity for AUA over AUG.


Assuntos
Archaea/genética , Códon , Isoleucina/genética , Biossíntese de Proteínas , RNA de Transferência de Isoleucina/química , Ribossomos/química , Archaea/química , Archaea/metabolismo , Isoleucina/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA de Transferência de Isoleucina/metabolismo , Ribossomos/metabolismo
13.
Cell Metab ; 14(3): 428-34, 2011 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-21907147

RESUMO

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.


Assuntos
Ciclo-Oxigenase 1/metabolismo , DNA Mitocondrial/química , Fibroblastos/metabolismo , Doença de Leigh/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas , RNA de Transferência de Metionina/metabolismo , Células Cultivadas , Criança , Ciclo-Oxigenase 1/genética , DNA Mitocondrial/genética , Fibroblastos/patologia , Heterozigoto , Humanos , Hidroximetil e Formil Transferases , Immunoblotting , Doença de Leigh/metabolismo , Doença de Leigh/patologia , Lentivirus , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , Biossíntese de Proteínas/genética , Análise de Sequência de DNA , Transdução Genética , Vírion
14.
Mol Microbiol ; 78(2): 506-18, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20807199

RESUMO

The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5'- and 3'-ends of 16S rRNA as well as maturation of the 5'-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.


Assuntos
Proteínas de Escherichia coli/metabolismo , Metaloproteínas/metabolismo , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Deleção de Genes , Metaloproteínas/genética , Dados de Sequência Molecular , Fatores de Iniciação em Procariotos/metabolismo , Ligação Proteica , Ribossomos/metabolismo , Alinhamento de Sequência
15.
Proc Natl Acad Sci U S A ; 107(7): 2872-7, 2010 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-20133752

RESUMO

Modification of the cytidine in the first anticodon position of the AUA decoding tRNA(Ile) (tRNA2(Ile)) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui, established its codon reading properties, used liquid chromatography-mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of tRNA2(Ile) adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the tRNA2(Ile) demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation C(+), for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis tRNA2(Ile) and in Sulfolobus solfataricus total tRNA, indicating its probable occurrence in the AUA decoding tRNA(Ile) of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG.


Assuntos
Anticódon/genética , Pareamento de Bases/genética , Citidina/química , Haloarcula marismortui/química , RNA de Transferência de Isoleucina/química , Agmatina/química , Cromatografia Líquida , Mathanococcus/química , Estrutura Molecular , RNA de Transferência de Isoleucina/genética , Sulfolobus solfataricus/química , Espectrometria de Massas em Tandem
16.
Mol Cell ; 35(2): 181-90, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19647515

RESUMO

Translation initiation of the second ORF of insect dicistrovirus RNA depends on an internal ribosomal entry site (IRES) in its intergenic region (IGR) and is exceptional in using a codon other than AUG and in not using the canonical initiator methionine tRNA. Studies in vitro suggest that pseudoknot I (PKI) immediately preceding the initiation codon occupies the ribosomal P site and that an elongator tRNA initiates translation from the ribosomal A site. Using dicistronic reporters carrying mutations in the initiation codon of the second ORF and mutant elongator or initiator tRNAs capable of reading these codons, we provide direct evidence for initiation from the A site in mammalian cells and, under certain conditions, also from the P site. Initiation from the A but not the P site requires PKI. Thus, PKI structure may be dynamic, and optimal IGR IRES-mediated translation of dicistroviral RNAs may require trans-acting factors to stabilize PKI.


Assuntos
Picornaviridae/genética , Biossíntese de Proteínas/fisiologia , Ribossomos/química , Códon de Iniciação , Códon de Terminação , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutação , Fases de Leitura Aberta , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência/fisiologia , RNA de Transferência de Metionina , Ribossomos/fisiologia , Transfecção , Regiões não Traduzidas , Proteínas Virais/genética , Proteínas Virais/metabolismo
18.
Methods ; 44(2): 129-38, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18241794

RESUMO

Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs and aminoacyl-tRNA synthetases. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA(Pro) with cysteine, but does not aminoacylate archaeal tRNA(Cys) with cysteine; (iv) identification and characterization of the AUA-decoding minor tRNA(Ile) in archaea; and (v) evidence that the archaeal minor tRNA(Ile) contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.


Assuntos
Aminoacil-tRNA Sintetases/análise , Eletroforese em Gel de Poliacrilamida/métodos , RNA de Transferência/análise , Animais , Archaea/metabolismo , Northern Blotting/métodos , Humanos , Concentração de Íons de Hidrogênio , Lisina/análogos & derivados , Lisina/biossíntese , Engenharia de Proteínas/métodos , Nucleosídeos de Pirimidina/biossíntese , RNA Bacteriano/isolamento & purificação , RNA de Transferência/isolamento & purificação , RNA de Transferência de Cisteína/biossíntese , RNA de Transferência de Isoleucina/metabolismo , RNA de Transferência de Metionina/metabolismo , Ureia
19.
Mol Microbiol ; 67(5): 1012-26, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18221266

RESUMO

Despite its importance in post-transcriptional regulation of polycistronic operons in Escherichia coli, little is known about the mechanism of translation re-initiation, which occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. To investigate translation re-initiation in Escherichia coli, we constructed a di-cistronic reporter in which a firefly luciferase gene was linked to a chloramphenicol acetyltransferase gene using a segment of the translationally coupled geneV-geneVII intercistronic region from M13 phage. With this reporter and mutant initiator tRNAs, we show that two of the unique properties of E. coli initiator tRNA - formylation of the amino acid attached to the tRNA and binding of the tRNA to the ribosomal P-site - are as important for re-initiation as for de novo initiation. Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation. In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation. Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Iniciação Traducional da Cadeia Peptídica , Fator de Iniciação 2 em Procariotos/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , RNA de Transferência de Metionina/metabolismo , Bacteriófago M13/crescimento & desenvolvimento , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fator de Iniciação 2 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/genética , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genética , Ribossomos/metabolismo
20.
RNA ; 14(1): 117-26, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17998287

RESUMO

Annotation of the complete genome of the extreme halophilic archaeon Haloarcula marismortui does not include a tRNA for translation of AUA, the rare codon for isoleucine. This is a situation typical for most archaeal genomes sequenced to date. Based on computational analysis, it has been proposed recently that a single intron-containing tRNA gene produces two very similar but functionally different tRNAs by means of alternative splicing; a UGG-decoding tRNA(TrpCCA) and an AUA-decoding tRNA(IleUAU). Through analysis of tRNAs from H. marismortui, we have confirmed the presence of tRNA(TrpCCA), but found no evidence for the presence of tRNA(IleUAU). Instead, we have shown that a tRNA, currently annotated as elongator methionine tRNA and containing CAU as the anticodon, is aminoacylated with isoleucine in vivo and that this tRNA represents the missing isoleucine tRNA. Interestingly, this tRNA carries a base modification of C34 in the anticodon different from the well-known lysidine found in eubacteria, which switches the amino acid identity of the tRNA from methionine to isoleucine and its decoding specificity from AUG to AUA. The methods described in this work for the identification of individual tRNAs present in H. marismortui provide the tools necessary for experimentally confirming the presence of any tRNA in a cell and, thereby, to test computational predictions of tRNA genes.


Assuntos
Códon , Haloarcula marismortui/genética , RNA de Transferência/genética , Acetilação , Anticódon , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA de Transferência/química
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